Phospholipase A2 (PLA2) represents a superfamily of intracellular and secreted enzymes that hydrolyze the sn-2 ester linkage of phospholipids. It has been well known that PLA2 plays important roles in many human diseases, including cardiovascular diseases, neurological disorders and cancer. Even though many methods (e.g. colorimetric, radioactive, fluorogenic) have been developed to monitor the activity of PLA2 there are many limitations for their uses, in particular, real-time continuous monitoring in living cells.
Recently Schultz and his coworkers described the PLA2 substrate based on fluorescence resonance energy transfer which appears to be a versatile quantitative real-time assay for PLA2 with fluorescence ratiometric analysis and selectivity for specific PLA2 in living cells. Agnew. Chem. Int. Ed., 2006, v. 45, pgs. 508-512. Schultz covalently attached NBD fluorophore as a donor at sn-1 position with nonhydrolyzable ether linkage and Nile Red at sn-2 position as an acceptor with ester bond (A).

While this substrate is reported to be useful for monitoring real-time activity of PLA2 in living cells, it has significant limitations due to the low photostability and low fluorescence quantum yield (QY) of the fluorophores (NBD and Nile Red) in particular for fluorescence imaging applications. In addition NBD and Nile Red are both enviroment-sensitive dyes, so the signal intensity and wavelength are biased by environmental factors.
Accordingly an objective of the present invention is to provide an improved PLA2 selective enzyme substrate with high fluorescence QY and greater photostability.